Monday, March 11, 2013

Schizophrenia, in a dish

by Kristin Sheppard

A few weeks ago my friend Ellen
posted a great blog about stem cells. She did a great job of explaining the
basics of stem cells and describing some of the possible therapies and maybe
even cures for various diseases that would accompany transplantation of these
cells under different conditions (and if you missed it, you can read her blog
post here).

Cal Poly CIRM students class of 2014

Over the last few weeks both Ellen
and I (and everyone else in our little cohort of first-year CIRM students) have
been tirelessly researching different labs, and the work that these labs are
doing in the field of stem cell technology, to try and find the perfect fit for
our upcoming internships. During my research I stumbled upon a whole new aspect
of stem cell research that until recently has been unexplored. A post-doc from
the Gage lab in the Laboratory of Genetics at the Salk Institute for Biological
Sciences presented it to me as “schizophrenia in a dish.” As crazy as it
sounds, this lab has been able to make a model of this disease by taking a very
small skin sample from patients with schizophrenia, turning those cells back
into stem cells (technically they would be called induced pluripotent stem
cells or iPSCs), and then differentiating the iPSCs into neurons. By culturing
these cells in a plate instead of an animal they have created a cellular model
of schizophrenia, in a dish.

So let’s take a step back for a
minute, and discuss what schizophrenia actually is. Schizophrenia affects
approximately 1% of the world population. It is a mental disorder characterized
by the experience of psychosis, a state where the individual loses contact with
reality. To be clinically diagnosed with schizophrenia, the individual needs to
satisfy the following requirements:

1)Have at least of the two following
symptoms for a significant period of time during a one-month period:

・Delusions

・Hallucinations
(either visual or auditory)

・Disorganized
speech

・Excessively
disorganized or catatonic behavior

・Negative
symptoms (which can include a reduction in speech or speech content, flat
affect, increased apathy, or social withdrawal)

2)Functioning far below their functional
level prior to the onset of symptoms.

3)Continuous signs of the disturbance for
at least six months, and at least one of those months include symptoms in their
full and active form.

So
if schizophrenia is a mental disorder diagnosed based on a person’s behavior
and perceptions, how can a bunch of cells in a dish be helpful? Although the
exact cause is unknown, schizophrenia tends to run in families, suggesting that
there is a genetic component. There have been several genes found to be associated with schizophrenia, but there is still no clear gene or
set of genes that cause the disease. That is where the cellular model comes in.
This model allows scientists to test molecular differences and differences in
electrophysiology between neurons from individuals with schizophrenia and
individuals without schizophrenia. Even better than that, this model
circumvents complications that come with current methods of studying this
disease in humans (which generally means a post-mortem analysis of an affected
individual’s brain). Common complications that accompany these post-mortem
studies include an unknown history of medical treatment for the disease (or
lack there of), a history of alcohol or drug abuse, and variations in the cause
of death. The cellular model is a living model with identical neurons from
living individuals who can likely identify their treatment history. This is
important because it allows for the direct comparison with neurons from
unaffected individuals to find molecular and physiological differences. It also
allows for the treatment of the affected neurons with different medications to
compare how these drugs affect genetically identical cells to help determine
what the best treatment is.

So
lets get back to what these researchers actually found. The initial findings,
although important, were not that exciting. They found that when they turned
the skin cells into stem cells, then differentiated them into the neural
progenitor cells (the precursors for neurons), and finally neurons there was
absolutely no difference between cells from individuals with schizophrenia and
cells from individual without schizophrenia. More specifically, there was no
difference in the differentiation process or pluripotency markers between cells
from schizophrenic individuals and cells from non-schizophrenic individuals.

After
these cells were allowed to become mature neurons, they tested the synaptic
connections between these neurons. The way that they did this was particularly
clever; they used a rabies virus. The rabies virus is known to spread between
neurons in an animal by moving through the synapses (or connections) between neurons. However,
in order to be able to use this to test the connectivity of the neurons, the
researchers have to be able to distinguish neurons that were infected directly
from the surrounding fluid and neurons that were infected through the synapse
with another neuron. In order to do this they modified the rabies virus so that
it is fluorescent red when viewed with a fluorescence microscope, and has to use
a specific receptor to be able to enter the neuron from the surrounding
fluid. Before
putting the modified rabies virus into the surrounding fluid, they modified the
neurons so that only some of them had the necessary receptor for the rabies
virus. The neurons with this receptor would appear fluorescent green when
viewed under the same microscope. This is important because you can tell which
cells were infected from the surrounding fluid (because they will be both red
and green) and which ones were infected through its connection to another
neuron (because those will be only red). They then compare the number of just
red cells to the number of red and green cells to determine the connectivity of
the neurons, a higher proportion of just red cells means that there is better
connectivity between those neurons. So these researchers found that neuron
cultures from individuals with schizophrenia had fewer just red cells than neuron
cultures from individuals without schizophrenia. This means that neurons with
schizophrenia have lower connectivity than neurons without schizophrenia. This
is a physiological difference that would not have been found without the use of
the cellular model.

Neurons infected with rabies virus. The image on the left is of neurons from non-schizophrenic individuals, image on the right is of neurons from a schizophrenic individual (Brennand, 2011).

They
also found that neurons from individuals with schizophrenia had fewer
projections out of the cell body than neurons from individual without schizophrenia.
This finding was consistent with previous observations made from post-mortem
studies on the brains of schizophrenic individuals. It is however important
that this was confirmed, because it is another reason why neurons from
schizophrenic individuals have lower connectivity. These projections are responsible for making the connections known as synapses with other neurons,
so fewer projections means fewer connections.

When
they measured the electrical signals being sent between these neurons they
found that there were no differences between neurons from schizophrenic
individuals and non-schizophrenic individuals in the characteristics of the
electrical signals or the ability of the neurons to send the signals. This
would indicate that these neurons are still able to communicate with each
other; it’s mainly a difference in the number of connections that allow
communication between neurons and which signals are being sent that
distinguishes schizophrenic neurons from non-schizophrenic neurons.

After
discovering the decreased connectivity, these researches tested the effects of
five common antipsychotic drugs and their ability to increase the connectivity
between these neurons. The drugs that they tested were Clozapine, Loxapine,
Olanzapine, Risperidone, and Thioridazine. They found that only Loxapine
significantly improved the neural connectivity while they were in the dishes.
This would imply that Loxapine is going to be the most effective medication for those
individuals, however they also admit that it is possible that they need to
adjust the doses or the timing of when they administer the other medications to
be able to see an effect. Although they may need to work out the dosages, this
method can be an effective way for screening for new drugs that will help to
treat or even cure the disease.

It's difficult to know all the side effects of a drug when they're not tested in a cellular model first

Finally,
using this cellular model, researchers found that there were 596 unique genes with
a 1.3 fold change in how many times each gene was read by the cells between the
schizophrenia neurons and the non-schizophrenic neurons. Only 13% of these
genes had been previously identified in publications to be associated with
schizophrenia. A whole 16% had been previously linked to schizophrenia through
post-mortem studies. And a whopping 25% had been previously implicated in
schizophrenia in one manner or another. This is very important because it
implies that there isn’t just one gene responsible, or even just a couple genes
responsible for causing this disease. In fact, the researchers think that as
this model is used to study more individuals with schizophrenia, it will be
found that there are countless different changes in the way an individual’s
cells will read their DNA. That sounds terrifying right? But the good news is
that they also predict that although there may be a ton of small changes, they
think there will be a pattern in what causes these changes, meaning that they
think there will be a handful of common pathways that are disrupted that leads
to all the countless small changes.

So let’s do a recap. This research team
from the Gage lab at the Salk Institute for Biological took skin cells from individuals
with schizophrenia and used stem cell technology to turn them into neurons.
They then compared these neurons to neurons that had been created in the same
way, but from skin cells of individuals without schizophrenia. They found that
neurons from schizophrenic individuals have a lower connectivity and fewer
projections from the cell body, which would contribute to the lower
connectivity. They also found that there are an amazingly large number of
molecular differences in how neurons from schizophrenic individuals read their
DNA, but they expect to find a common pathway that is causing all these small
changes as more individuals are studied in this manner. And finally they found
that this model can be used as a way to screen new medications for their effectiveness
in treating the physical aspects of this disease.

Now all of this is really cool and
all, but why should anyone except for scientists and those who are affected in
one way or another by schizophrenia care?
If this cellular model can work for such a complex disease as
schizophrenia, it should also be able to work to study, and maybe even cure, equally
complex diseases that are more common such as autism, multiple sclerosis, or even
Parkinson’s disease. In this unique way stem cells now present the potential to help understand, and maybe even cure, diseases with currently unknown causes.

1 comment:

Very interesting! I had no idea that stem cell technology had advanced to the level that cell could be induced into a pluripotent state and then differentiated to a target cell type, that's amazing. It's another tool that could help determine the overall model of schizophrenia, a complex and sad disease. Great blog Kristin.